JOHN W. CHURCHMAN 23 



decolorize them. The demonstration that bacteria contain an abundance of nucleo- 

 protein appears to explain their affinity for basic coal-tar dyes, and there are many 

 grounds for the belief that— perhaps in the majority of cases — staining is due to a 

 weak combination between nucleoprotein and basic dye, decoloration being due to 

 dissociation of the dye-protein compound. Bacteria certainly behave in their staining 

 reactions as if composed largely of protein. They were formerly thought to consist 

 entirely of nucleus and cell wall without cytoplasm. Whether this be true or not, one 

 other element in bacterial cells is of great — perhaps of prime — importance in many 

 staining phenomena, namely, lipoids. Bacteria are known to contain fats and lipoids 

 in varying amounts which, because of the marked effect on surface tension, would for 

 purely physical reasons tend to become concentrated at the periphery of such a col- 

 loidal system as the bacterial protoplasm. Whether or not there is a morphologically 

 distinct limiting membrane, we can reasonably assume that the surface of the bac- 

 terial cell is potentially lipoid. The presence of unsaturated fatty acids in the lipo- 

 protein of the bacterial cell is thought by many to explain the mechanism of the gram 

 stain. 



The amount of dye taken up by bacteria and the firmness with which it is held 

 doubtless depends in large part on the H-ion concentration of bacterial protein. Since 

 the pH of bacterial protein may change with age it might be expected that the stain- 

 ing characteristics of bacteria would change, and it is of course well known that this 

 does occur; the difference between the gram behavior of young and old cultures may 

 be cited as an example. 



The chief physical factor of the staining process — emphasized by advocates of the 

 physical hypothesis to explain the phenomena of dyeing, and which must in any event 

 be taken into consideration — is the process of adsorption of the dye by the bacterial 

 substance after it has passed through the wall membrane by osmosis. Adsorption is 

 the property possessed by a solid body of attracting to itself by physical means from a 

 surrounding solution certain compounds or ions present in that solution. In the case 

 of the dyes it is assumed that, when once adsorbed, they remain in the stained tissue 

 in solid solution. There are certain facts which point strongly to a physical explana- 

 tion of staining. For example, there is no evidence of the formation of a new substance 

 when tissue is stained; the colored tissue merely takes on one of the characteristics of 

 the dye (color). It is usually possible to extract all or nearly all the dye by immersion 

 in water or alcohol. Furthermore, tissue never removes all the stain from the dye solu- 

 tion, no matter how dilute. These facts must be borne in mind, and physical processes 

 must be thought of as playing an important role. But more and more evidence is ac- 

 cumulating to indicate that strictly chemical processes are also concerned. 



THE GRAM STAIN 



Dyes are used in bacteriology for four purposes: (i) to make organisms visible, 

 (2) to display their structure, (3) to reveal their chemical nature, and (4) to influence 

 their growth. It is well known that most bacteria are stained easily and a few with 

 difficulty, and that certain parts of the bacterial cell (the spores, capsules, and flagel- 

 la) are not stained at all by ordinary methods. However, with the exception of the 

 acid fast group, the vast majority of bacteria behave very much alike toward simple 



